Eukaryotic cell division proceeds through a highly regulated event, i.e. the cell cycle, comprising consecutive phases termed G1, S, G2 and M. Disruption of the cell cycle or of the cell cycle control mechanisms can result in cellular abnormalities or disease states such as cancer. The disruption of cell cycle control can be due to multiple genetic changes that transform growth-limited cells into highly invasive cells that are unresponsive to normal control of growth. Transition of normal cells into cancer cells can arise though loss of correct function in DNA replication and DNA repair mechanisms. All dividing cells are subject to a number of control mechanisms, known as cell-cycle checkpoints, which maintain genomic integrity by arresting or inducing destruction of aberrant cells. The investigation of cell-cycle progression and cell-cycle control is therefore of significant interest for revealing the molecular mechanims underlying cell cycle proliferation, as well as its disregulation in form of, for example, cancer. Consequently, revealing the principles of cell cycle progression is a needful help in designing anticancer drugs (as described in Tyson and Novak, Curr. Biology 18, R759-R768, 2008).
The major events of the eucaryotic cell cycle—such as DNA replication, mitosis, and cytokinesis—are triggered by a well-defined and sophisticated cell cycle control system. A major component of this cell cycle control system is a family of protein kinases known as cyclin-dependent kinases (Cdks). The oscillating activity of these kinases directly correlates with the initation or regulation of cell cycle progression, wherein the cyclical changes in Cdk activity in turn are controlled by a variety of enzymes and other proteins. The most important Cdk regulators are proteins known as cyclins which undergo a cycle of synthesis and degradation in each cell cycle. That is, the cyclin dependent kinases associate successively with different cyclins to trigger the different events of the cell cycle, and the activity of the Cdks is usually terminated by cyclin-dependent proteolytic degradation.
Cell cycle control crucially depends on at least two distinct enzyme complexes that act at different times in the cycle to cause the proteolysis and thus the inactivation of key proteins of the cell cycle control system. Most notably, cyclin-Cdk complexes are inactivated by regulated proteolysis of cyclins at certain stages of the cell-cycle, wherein the destruction of cyclins occurs by a ubiquitin-dependent mechanism in that an activated enyzme complex recognizes specific amino acid sequences on the protein to be degraded and attaches multiple copies of ubiquitin to it, thereby marking the protein for complete destruction by the 26S proteasome. Ubiquitination is a covalent modification which involves the formation of an isopeptide bond between the carboxy-terminus of ubiquitin and the ε-amino group of a lysine residue within the acceptor protein.
The accurate determination of the cell cycle status is a key requirement for investigating cellular processes that affect the cell cycle and/or cell cycle progression, for example, in the context of various biological processes including, e.g. wound healing, cancer, or development. Furthermore, the accurate determination and control of cell cycle progression has an important impact on all aspects of regenerative medicine which indispensably relies on the use of embryonic stem (ES) cells. Embryonic stem (ES) cells are characterized by their ability to develop into all cell types of the body. However, remarkably little is yet known about the exact factors that make them differentiate into a specific type of cell. The analysis of cell cycle progression on the basis of the proliferative and developmental potential of embryonic stem (ES) cells thus promises successful strategies for transplantation therapies ranging from, e.g., heart disease to Parkinson's disease to leukemia due to the almost unlimited supply of specific cell types.
Cell cycle progression is tightly regulated by the defined temporal and spartial expression, localisation and destruction of a number of cell cycle regulators which exhibit a highly dynamic behavior during the cell cycle. For example, at specific stages of the cell cycle, some proteins translocate from the nucleus to the cytoplasm, or vice versa, thereby marking and controlling the transition from one phase of the cell cycle to another, e.g. the transition from G2 phase to mitosis or its exit from mitosis. Others are expressed only at specific phases of the cell cycle and are then rapidly degraded.
The mechanisms and control elements which regulate the temporal expression and destruction of protein factors with a key role in cell cycle progression have been elucidated in a number of studies (see, for example, Nurse, P., Cell 100 (1), 71-78, Jan. 7, 2000). More recently, the visualization of spatiotemporal dynamics of eukaryotic cell cycle progression including the visualization of proteins that mark cell cycle transitions has come into focus. For example, several cell-cycle markers that identify the S phase and the subsequent transition to G2 in live cells have been developed by fusing fluorescent proteins to nuclear proteins such as proliferating cell nuclear antigen (PCNA), DNA ligase I, or the C terminus of helicase B (as described, for example, in Easwaran et al., Cell Cycle 4, 453-455, 2005). However, since identification of cell-cycle transitions requires the detection of subtle and often minute changes in the distribution pattern and intensity of fluorescence signals, these markers are not suitable to track cell cycle phase transitions with high contrast.
Determination and modulation of cell cycle progression have important implications for all aspects of stem cell biology and regenerative medicine. Current strategies in the field focus on re-induction of proliferation in postmitotic cells or use of stem cells as a source for cell replacement therapies. The most widely used approach for the identification of proliferating cells is based on staining of fixated cells with typical proliferation markers such as Ki-67 PCNA or pHH3. This, as well as the use of thymidine analogons such as BrdU, CldU or IdU, is prone to a number of artefacts, one of the most common being the labelling of cells undergoing endoreduplication, acytokinetic mitosis or DNA repair (as described, for example, in Breunig, J. J. et al., Cell Stem Cell 1, 612-627, 2007). Endoreduplication is defined as continuing rounds of DNA replication without karyokinesis or cytokinesis (as described in Storchova, Z. and Pellman, D., Nat. Rev. Mol. Cell. Biol. 5, 45-54, 2004; Brodsky, W. Y. and Uryvaeva, I. V., Int. Rev. Cytol. 50, 275-332, 1977), whereas acytokinetic mitosis is karyokinesis without cyotkinesis. Both are common processes during development and under pathological conditions in tissues such as cardiac muscle, liver, or uterine decidua. The resulting polyploidity causes false positives in proliferation assays and an overestimate of dividing cells.
During postnatal heart growth and development cardiac muscle cells undergo acytokinetic mitosis resulting in binuclear cells (as described in Soonpaa et al., Am J Phys, 271, H2183-9, 1996), followed by endoreduplication (as described in Storchova, Z. and Pellman, D., Nat. Rev. Mol. Cell. Biol. 5, 45-54, 2004); these cells are thought to be incapable of performing cytokinesis (as described in Pasumarthi, K. B. and Field, L. J., Circ. Res. 90, 1044-1054, 2002). Current approaches to assess cardiac proliferation cannot provide detailed insight into variations of the cell cycle rendering the estimate of cardiac muscle renewal inaccurate. In fact this and additional caveats are likely to underlie the substantial controversy about proliferating cardiac muscle cells reported for intact and diseased or infarcted hearts (as described in Soonpaa, M. H. and Field, L. J., Circ. Res. 83, 15-26, 1998; Hsieh, P. C. et al., Nat. Med. 13, 970-974, 2007; Beltrami, A. P. et al., N. Engl. J. Med. 344, 1750-1757, 2001). The observation of cytokinesis consisting of a contractile ring and the appearance of a midbody prior to daughter cell separation would be the only definitive proof for proliferative activity in cardiomyocytes.
Visualization of the cell-cycle transition from G1 to S phase in live cells has recently been described by the development of a dual-color imaging system in which the inversely oscillating human E3-ligase substrates Cdt and Geminin have been fused to red- and green-emitting fluorescent proteins, respectively (as described in Sakaue-Sawano et al., Cell 132, 487-498, 2008). This so called “Fucci” system (fluorescent ubiquitination-based cell cycle indicator), which employs the parallel use of two individual reporter constructs, allows for the monitoring of structural changes and cell cycle dynamics of individual cells by labeling G1 phase nuclei red and S/G2/M phase nuclei green.
WO 03/031612 describes a nucleic acid reporter construct comprising a nucleic acid sequence encoding a detectable live cell reporter molecule under the control of at least one cell cycle phase-specific expression control element and a destruction control element, as well as methods for determining the cell cycle position of a cell related thereto.
There is always need for an improved method of visualizing and analyzing cell cycle progression in eucaryotic cells, for example, for systems that allow the identification of cell division versus mere cell cycle variation. A promising approach to unequivocally prove cell division is the direct visualization of the contractile ring and midbody occurring in the late mitotic (M) phase of the cell cycle. The contractile ring assembles equatorially at the cell cortex and constricts the cell membrane, thereby forming two daughter cells. At the same time the midbody, which is a microtubule rich structure located in a small cytoplasmic bridge between the daughter cells, forms and their separation is completed by its cleavage.
The identification and modulation of proliferating cells is of high relevance for stem cell research and regenerative medicine. However, current approaches to quantify cell proliferation are inaccurate in tissue types such as heart muscle cells displaying variations of the cell cycle namely acytokinetic mitosis and endoreduplication. To overcome these limitations, the present invention provides an in vivo reporter system using a protein with a wild-type destruction signal fused to a reporter protein for high resolution of the M-phase. This enables the visualization of cytokinesis and midbody formation as hallmarks of cell division in virtually all cell types and organs of transgenic mice. Furthermore, the present invention enables cell division, acytokinetic mitosis and endoreduplication to be distinguished. Thus, this new assay allows the monitoring and quantitation of cell proliferation and division in vitro and in vivo even in tissue types prone to DNA repair and variations of the cell cycle.